Obesity among adults and children has become increasingly prevalent in almost all industrialized countries. According to the National Institute of Health, over the past two decades the incidence of obesity has increased more than 50 percent. The primary causes of obesity are not precisely understood, although social, dietary, and genetic factors are thought to be involved. Obesity represents a major risk factor for cardiovascular diseases, diabetes, osteoporosis, and atherosclerosis. It is believed that a 10% reduction of weight corresponds to a 20% reduction in coronary disease incidence, whereas a 10% increase in weight is associated with a 30% increase in coronary disease incidence [“Obesity: Preventing and Managing the Global Epidemic” in WHO Obesity Technical Report Series 894. World Health Organization, Switzerland, 2000; Rose. Nutr Metab Cardiovasc Dis 1991 1:37–40; Ashley and Kannel. J. Chronic Dis. 1974 27:103–114]. Thus, reduction of obesity is of major importance to improve general health.
The term “obesity” refers to a state of excess body fat mass. This body fat mass is comprised of adipocytes. The primary function of adipocytes is to store excess energy in the form of triacylglycerol, and to release this stored energy as free fatty acid in times of increased energy need. Triacylglycerol is stored within adipocytes in the form of lipid droplets. The lipid droplets are composed of a hydrophobic core of neutral lipids (such as triacylglycerol) surrounded by a phospholipids monolayer, in which protein is embedded. Upon increased demand for energy (e.g., during fasting or exercise), this stored triacylglycerol is hydrolyzed to yield glycerol and free fatty acids. These hydrolysis products are release into the blood stream. The free fatty acids are then taken up by, and used for the generation of ATP in, a wide variety of tissues.
The balance of energy storage versus release by adipocytes is tightly regulated. For example, the anti-lipolytic hormone insulin stimulates the assembly of triacylglycerol lipid droplets in adipocytes. Conversely, lipolytic hormones, such as epinephrine, norepinephrine, catecholamines, and Tumor Necrosis Factor alpha (TNFα), activate the hydrolysis of triglycerides. Reduction of the body fat mass occurs when pro-lipolytic effects predominate, such that triacylglycerol stores are hydrolyzed and adipocyte mass decreases.
Two key players in the regulation of the adipocyte lipolytic pathway are Hormone Sensitive Lipase (HSL) and perilipin.
The HSL enzyme catalyzes the rate limiting step of triglycerides hydrolysis. Lipolytic hormones activate HSL by stimulating its enzymatic activity and/or promoting its translocation from the cytoplasm to the periphery of the lipid droplet. This activation is thought to be mediated, at least in part, by phosphorylation of HSL by cAMP-dependent protein kinase (PKC).
Perilipin is the most abundant protein of the adipocyte lipid droplet. Up to four different isoforms of the perilipin protein may be generated by alternate splicing of a transcript produced from a single perilipin gene (Servetnick, et al. J. Biol. Chem. 1995 270:16970–16973). Perilipin protein is located at the periphery of the lipid droplet (Greenberg, et al. J. Biol. Chem. 1991 266:11341–11346), where it is thought to act as a barrier that precludes access of HSL to the stored triglycerides (Clifford, et al. FEBS Letters 1998 435:125–129; Souza, et al. J. Biol. Chem. 1998 273:24665–24669; and Brasaemle, et al. J. Biol. Chem. 2000 275:38486–38493). Lipolytic hormones antagonize the barrier function of perilipin, thereby allowing HSL to hydrolyze the lipid droplet triglycerides (Clifford, et al. J. Biol. Chem. 2000 275:5011–5015). This antagonism may be mediated, in part, by phosphorylation of perilipin by cAMP-dependent protein kinase (PKC).
The link between perilipin and HSL, and the importance of perilipin in the regulation of triglycerides storage, has been shown by a number of studies. Ectopic expression of perilipin in fibroblastic 3T3-L1 pre-adipocyte cells in vitro caused the formation of intracellular lipid droplets (Brasaemle, et al. J. Biol. Chem. 2000 275:38486–38493). These cells stored significantly more triacylglycerol that control cells. This increased storage was due to decreased hydrolysis of triacylglycerol. Ectopic expression of perilipin in 3T3-L1 cells also blocked the ability of TNFα to stimulate lipolysis (Souza, et al. J. Biol. Chem. 1998 273:24665–24669).
Mice lacking the perilipin gene are more muscular, have less fat, are resistant to diet-induced obesity, and have higher energy expenditure (Martinez-Botas, et al. Nature Genetics 2000 26:474–479; Tansey, et al. Proc Natl Acad Sci USA. 2001 98:6494–6499). In these mice, lipid droplet triglycerides are broken down as soon as they are made. These effects are related to the constitutive activation of HSL observed in these mice (Martinez-Botas, et al. Nature Genetics 2000 26:474–479). This result in mice establishes that disruption of perilipin function results in a decrease in fat mass, and prevents obesity.
Therefore, HSL and perilipin are attractive targets for drugs to treat obesity. Treatments which can activate HSL activity and simultaneously disrupt the perilipin shell of lipid droplets will promote body fat mass loss by stimulating lipolysis. The discovery of a novel pharmaceutical composition which has these effects, thus represents a significant advance in the treatment of obesity.
A number of dietary supplements and pharmaceuticals have been introduced for the promotion of fat loss (see, for example, U.S. Pat. Nos. 5,626,849; 5,804,596; 5,783,603; and 6,340,482). Most of the currently available pharmaceuticals for weight loss work by suppressing appetite via central mechanisms (Stunkard. Life Sci. 1982 30:2043–2055; Weiser, et al. J Clin Pharmacol. 1997 37:453–473). See, for example U.S. Pat. No. 5,783,603, which concerns the use of potassium hydroxy-citric acid to suppress appetite and reduce fatty acid synthesis.
Certain compounds have been disclosed that are able to induce weight loss by mechanisms other than appetite suppression (e.g., through stimulation of the peripheral metabolic rate of adipose tissue). For example, U.S. Pat. Nos. 4,451,465; 4,772,631; 4,977,148; and 4,999,377 disclose compounds possessing thermogenic properties which cause few or no deleterious side effects (such as cardiac stimulation) at the suggested dose. Another compound, Orlistat, blocks absorption of ingested fat by inhibiting pancreatic lipase.
The most commonly used natural supplements for promotion of fat loss include hydroxycitric acid (U.S. Pat. No. 5,626,849), L-carnitine, ma huang (ephedrine) and green tea, forskolin, and citrus extract (U.S. Pat. No. 6,340,482). In addition, chromium picolinate is sold as an ingredient in “fat burner” formulas (Evans. Int J Biosocial Med Res. 1989 11:163–80). However, subsequent clinical studies have failed to demonstrate any significant effect of chromium picolinate, hydroxycitric acid, forskolin or other natural dietary supplements on body fat mass reduction (Hasten, et al. Int J Sport Nutr 1992 2:343–50; Clancy, et al. Int J Sport Nutr. 1994 4:142–53). A well-controlled study found no difference in the magnitude of body fat mass or lean body mass between those who took the above mentioned dietary formulas and those who took placebo (Trent and Thieding-Cancel. J Sports Med Physical Fitness 1995 35:273–80).
The ineffectiveness of prior described dietary formulas in the treatment of obesity and promotion of fat mass reduction may be related to the fact that none simultaneously disrupts the perilipin shell of lipid droplets and stimulates HSL activity.
The essential ingredients of the novel composition have been used individually for treatment of prevention of various health disorders, and thus have a long history of safe use in humans. However, neither of these components has been used for the treatment of obesity and promotion of fat loss in mammals, and in particular in humans.
Dihydroquercetin-3-rhamnoside and aglycon dihydroquercetin (see FIG. 1) are flavonoids found commonly in many different plant species, such as Grape stem (Vitis vinifera) and the leaves of Rhododendron caucasicum and Larix siberica. The dihydroquercetins in Engelhardtia chrysolepis tea, known as kohki tea, have been consumed in Vietnam, Japan and other Asian cultures for centuries (Kasai, et al. Chem. Pharm. Bull. 1988 36:4167–4170; Igarashi, et al. Biosci. Biotec. Biochem. 1996 60:513–515; Mizutani, et al. Nippon Shokuhin Shinsozai Kenkyukaishi. 1998 1:51–64). Dihydroquercetins possess superior antioxidant activity to suppress lipid peroxidation and protect against the destructive affects of free radicals (Haraguchi, et al. Biosci Biotechnol Biochem 1996 60:45–48). Dihydroquercetins also stabilize blood vessels and protect against factors that cause atherosclerosis and cardiac, hepatic, and bronchio-pulmonary diseases. In addition, dihydroquercetins possess anti-allergy and anti-inflammatory activity (Tiukavkina, et al. Nutrition, Moscow 1997 6:12–15; Teselkin, et al. Biofizika. 1996 41:620–624; Tiukavkina, et al. Nutrition 1996 2:33–38), and protect the liver from toxins (Xu, et al. Eur J Pharmacol 1999 377:93–100). Dihydroquercetins have been approved in Russia as a medical preparations for use in antioxidant and capillary protective preparations (Russian National Pharmacopoeia Article 42-2398-94, approved on Jul. 29, 1996).
Dihydroquercetins are known to inhibit HMG-CoA reductase, a key enzyme in cholesterol synthesis (Chen, et al. Zhonghua Yi Xue Za Zhi, Taipei 2001 64:382–387) and lower plasma triglycerides levels (Igarashi, et al. Biosci. Biotec. Biochem. 1996 60:513–515; Mizutani, et al. Nippon Shokuhin Shinsozai Kenkyukaishi 1998 1:51–64). However, these effects of dihydroquercetins have not been associated with promotion of weight loss or fat reduction. This lack of functional association is consistent with the activity of other compounds used for the treatment of hypercholesteremia (e.g., statins), which reduce cholesterol and/or triglycerides levels without promoting fat loss. Thus, dihydroquercetins in isolation are not sufficient to effectively treat obesity and promote weight loss.
Plants of the genus Aralia are a natural source of the triterpene saponins known collectively as aralosides or elatosides. The terms “aralosides” and “elatosides” are derived from the scientific names of the Aralia plants from which these triterpene saponins may be derived (e.g., Aralia elata). These aralosides (or elatosides) have been shown to constitute the active ingredients of medicinal Aralia preparations. Eleven major triterpene saponins designated as aralosides A, B, C, D, E, F, G, H, I, J, and K have been isolated from Aralia (Yoshokawa, et al Chem. Pharm. Bull. 1994 43:1878–1882). The major active triterpene saponins of medical Aralia preparations are aralosides A, B, C, and D (see FIG. 2).
Based on various clinical studies, Aralia tincture has been included in the Russian State Pharmacopoeia XI under the name “Saporali” (Sokolov. Pharmacology and Chemistry, Moscow Science Publishing, Russia, 1968). Aralosides stimulate the central nervous and immune systems, possess anti-stress properties, and protect against unfavorable environmental conditions such as hypoxia or viral infections. Aralosides also improve long term memory (Sedykh, et al. “The Influence of the Aralia mandshurica tincture, animalon or their combination for memorization of texts,” New Medicinal Preparations from Plants of Siberia and Far East. Tomsk State University, Russia: 1986. p. 132). Furthermore, aralosides reduce ethanol absorption (Yoshikawa, et al. Chem Pharm Bull. Tokyo 1993 41:2069–2071; Yoshikawa, et al. Chem Pharm Bull. Tokyo 1996 44:1915–1922); possess analgesic properties (Okuyama, et al. Chem Pharm Bull. Tokyo 1991 39:405–407); and reduce blood sugar levels (Yoshikawa, et al. Chem Pharm Bull. Tokyo 1996 44:1923–1927).
It has now been discovered that a combination of dihydroquercetins and aralosides is effective for treating obesity.